Summary: Ex-vivo expansion of CD34+ hematopoietic stem cells (HSC) is currently based on the use of "early acting" cytokines which are thought to amplify hematopoietic progenitor cells (HPC) formation. A major obstacle in attaining this objective drives from the pleiotropic nature of cytokines, in that they support proliferation and subsequent differentiation of HPC as well. Since differentiation correlates inversely with pluripotency, the effectiveness of cytokine-treated HSCs to support hematopoietic reconstitution could be compromised. Identifing the key elements influencing the decision of CD34+ cells to proliferate versus differentiate could have valuable implications for devising strategies to favor selective expension of HSCs. Using the CD34+ myeloid leukemic M1 cells as a model system, we are studying the regulatory mechanisms underlying the balance between proliferation and differentiation program in M1 cells. Interleukin-6 (IL-6) stimulateds the terminal differentiation program in M1 cells, a process characterized by sequential expression of early and late gene products.Treating M1 cells with interferon gamma (IFNg), on the other hand, induces the selective expression of early gene markers such as FcgRI and Ia antigen. Coculturing M1 cells with both IL-6 and IFNg however, leads to a time-dependent inhibition of the expression of late gene products, such as inducible nitric oxide synthase (iNOS) stimulated with IL-6. Recently, we have observed that the inhibitory effect of IFNg is not restricted to expression of iNOS alone but is indicative of a generalized inhibition of myeloid differentiation itself. IL-6-induced expression of lysozyme and of the receptor for M-CSF, c-fms, also considered an early myeloid marker, is markedly inhibited by IFNg. Phagocytic activity and acquisition of morphological specialization in M1 cells stiumulated with IL-6 are similarly impaired by coexposure with IFNg, with a large majority of cells still retaining an undifferentiated blast cell-like morphology, and suggestive of a block in exit from the cell cycle. Given this finding, we are currently examining the regulation of CD34+ expression M1 cells, since IL-6 stimulated exit from the cell cycle is known to be accompanied by downregulation of this cell surface antigen, and CD34+ has been used to define the potential for self-renewal in HSCs.